96 well plate reader problems

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96 well plate reader problems

Postby MediaFlak » Jul 30 2010 10:28 pm

Hi everyone,
I am running an assay with samples, completely empty wells, and blanks containing only medium.
I know that the machine sensitivity is going whacko because the reading keeps fluctuating, even for the empty wells and the blanks!

The first day it was around 4000, then 2000, then 2500, now up to 3000 for the empty wells. Proportional changes were made for the blanks and the other wells as well.

Can anyone help me with this dilemma?
Thank you.
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Re: 96 well plate reader problems

Postby CrowSan » Aug 02 2010 3:03 pm

There is always a "natural" fluctuation when reading fluoresesnce etc on a plate reader.
The number you get from the plate reader is a "relative" value and the machine will normally "turn up" the signal if the signal is weak (like turning up the sound or brightness on a TV) and there is no strong signal - this generally occurs if you are using optimal gain or setting the gain from a well. It is not unusual to get different numbers each time you use it - but the no's should be proportional to each other (ie one day well A1 with sample will be 10 000 and the blank will be 1 000, the next day A1 may be 15 000 and the blank 1 500 etc). You could try setting a "gain" value rather than using optimal or gain from a well and see if this smooths them out a bit but as long as the values are proportional don't worry about it. Hope that made sense :)
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Re: 96 well plate reader problems

Postby MediaFlak » Aug 02 2010 11:27 pm

Thanks CrowSan.

What should I do if some values were not proportional?

A few of my wells went down while the rest of the wells went up, vice versa...
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Re: 96 well plate reader problems

Postby CrowSan » Aug 03 2010 2:49 pm

That is a different problem which I have only really noticed when trying to get fluorescene at the extreme range of sensitivity. For example whan I was trying to detect DNA fluoresence on a standard curve using Cyquant (a DNA dye from invitrogen) from as little as 10 cells per well to 200 cells (lowest sensitivity for cyquant is 50 cells according to the manufacturer). then I noticed that if I did multiple reads some signals went up/down. The problem here was my signal to noise ratio. I also found that just cleaning the plate (with ethanol on a bottom-read) had an effect on some of the signals!
There are a number of reasons why you might experience fluctuations between well to well:

i) As mentioned above your signal to noise ration is to low.
ii) The laser on your platereader is going and causing random power fluctuations (unlikely as you would see more problems in more than a few wells!).
iii) The samples you are reading are non-homogenous and the laser is detecting fluorescence from "clumps" that are drifting past the laser/detector.

Things you can try:
a) There is not much you can do if it is option i) above except try to increase the amount of "fluorescent substrate" you have per well. Sometimes using V-shaped wells help to concentrate sample to increase signal. Using "black walled" 96 well plates will also help to stop "signal leakage" between wells.
b) If you are doing a "bottom read" (this is not kinky way of divining the future but means that the spectroph. detectors are measuring the fluorescence from underneath the wells) try cleaning the base of the plate with 70% EtOH to make sure no small particles are auto-fluorescing (such as lint from paper towels etc). This is normally only a problem with weak signals. Also try doing a "top -read" (if your spec can do both) and see if this helps.
c) If it is the lasers or a problem with the detectors then your machine will need a service. Before invoking that expense you might want to set up some kind of standard, serial dilution curve on a plate (of whatever you are trying to detect) and see if you get a good linear curve out of the results. Remember as the spec measures "relative fluorescent units" you will need some kind of known standard on the plate anyway to make any qualative interpretation of your results and to be able to compare results from one plate to another.
d) If you have a low signal to noise ratio (and you know you have a lot of substrate present and thus a strong signal) it may be that you are getting background from the media. At some wavelenths the presence of DMEM can cause havok with the readings - I routinely avoid having any media present in the wells as it can generate some funny shapes in a standard curve! This is especially true for most of the common DNA dyes (Hoerscht, DAPI, Cyquant).

Unfortunately not being there in person and knowing what exactly you are doing I am not sure i can be much more help!
It sounds from what I've read (and this is just a guess) is that you have a poor signal to noise ratio that is either being caused by a auto-fluorescent background component (e.g. DMEM), a weak signal (caused by low fluorescent substrate) or both. Worst comes to the worst contact the manufacturer of the spec and see what they say (and if you have a service contract get them in to "tune" the machine up - and you can show them your problem at the same time).
Hope this has been a small help!
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Re: 96 well plate reader problems

Postby MediaFlak » Aug 05 2010 1:54 am

That has been a tremendous help.

Turns out I made a mistake in tuning the autosensitivity each time I did it. Apparently you are supposed to do autosensitivity the first time then repeat later on without that setting.

Thank you again!
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Re: 96 well plate reader problems

Postby CrowSan » Aug 05 2010 7:59 am

heh...I'm glad you got it sorted. :)
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